Page 273 - PC2019 Program & Proceedings

PLANT CANADA 2019

P119. Investigating the role of BKNs in pollen-stigma interactions
2
*1
Geng, B. ; J. Doucet ; D. Goring 1
1 University of Toronto
2 U of T

The Brassicaceae, are a family of plants that contain many scientifically as well as economically
important plants. Brassicaceae have “dry stigma”, meaning that the papillae lack surface secretions and
allows the plants to have greater control over the early post-pollination stages, with only compatible
pollen grains receiving water from the stigma, this facilitates pollen germination and fertilization. The
basal compatible response pathway in the stigma is initiated upon recognition of compatible pollen,
followed by exocyst-mediated transduction of secretion from the stigma towards the pollen, and this
pathway remains largely elusive. A previous attempt to identify candidate genes in the compatible pollen
signal transduction pathway led to the discovery of pseudokinases, BRASSIKIN1 (BKN1) and BKN2. It
was also discovered that BKN orthologous are found throughout the Brassicaceae family. The BKN loci
between different A. thaliana ecotypes as well as different Brassicaceae species differ. A. thaliana
ecotype Col-0 has a truncated BKN1 caused by a premature stop codon, but A. thaliana ecotype Hh-0 as
well as A. lyrata have fully intact BKN1 and 2 genes. For this project, BKN1 and BKN2 mutants will be
generated in Hh-0 and A. lyrata using the CRISPR-Cas9 system. As well, overexpression BKN1
constructs will be transformed into Col-0. Pollen response characterization assays will be performed to
further understand the potential importance of the BKN genes in the compatible pollen response pathway.

Betty Geng (b.geng@mail.utoronto.ca)

TOPIC 12: Diagnostic Tools Applied to Crop Production
(Poster P120)

P120. Laboratory testing of qPCR assays designed in silico reveal promising results to rapidly
identify phytopathogenic Tilletia species.
*1
2
1
2
1
Tremblay, E. ; D. Shearlaw ; H. Nguyen ; G. Bilodeau ; S. Hambleton
1 Agriculture and Agri-Food Canada
2 Canadian Food Inspection Agency
Tilletia species are grass fungal pathogens of concern, especially for grain-exporting countries like
Canada, because some cause diseases on wheat. Among them, Tilletia indica (Karnal bunt) and T.
controversa (dwarf bunt) are subject to quarantine regulations and therefore are highly unwanted in areas
that are free of those species. Both pathogens are closely related to un-regulated species: T. indica to T.
walkeri and T. controversa to T. caries or T. laevis. There is a need for more rapid and sensitive
diagnostic tools compared to the morphological methods currently used by regulatory agencies. Recent in
silico comparative genomics analyses identified sets of candidate qPCR primers and probes targeting
single copy genes that are species-specific. These markers have been tested in the laboratory against DNA
from pure culture isolates for nine species: T. indica, T. walkeri, T. asperifolia, T. barclayana, T. fusca, T.
rugispora, T. laevis, T. controversa, and T. caries. Although some assays showed differing levels of
cross-reactivity, one for T. walkeri and one for T. indica were highly specific with promising sensitivity
values (30-200 fg). The next steps will be to increase sensitivity by optimizing the parameters and
confirm efficacy by testing against Tilletia-infected field samples. The validated assays will help improve
diagnostic throughput, speed and accuracy for these agriculturally important pathogens.

Emilie Tremblay (emilie.tremblay@canada.ca)

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